Biomaterials and Tissue Engineering Strategies for Conjunctival Reconstruction and Dry Eye Treatment.

The ocular surface is a component of the anterior segment of the eye and is covered by the tear film. Together, they protect the vital external components of the eye from the environment. Injuries, surgical trauma, and autoimmune diseases can damage this system, and in severe cases, tissue engineering strategies are necessary to ensure proper wound healing and recovery. Dry eye is another major concern and a complicated disease affecting the ocular surface. More effective and innovative therapies are required for better outcomes in treating dry eye. This review focuses on the regenerative medicine of the conjunctiva, which is an essential part of the ocular surface system. Features and advances of different types of biomolecular materials, and autologous and allogeneic tissue grafts are summarized and compared. Specifically, vitrigel, a collagen membrane and novel material for use on the ocular surface, offers significant advantages over other biomaterials. This review also discusses a breakthrough microfluidic technology, "organ-on-a-chip" and its potential application in investigating new therapies for dry eye.

INTRODUCTION

The eye is the organ that enables us to perceive the world around us. The ocular surface is an essential component of the visual system, as it protects the vital parts of the eye and provides the appropriate refracting surface for light rays entering the eye. The ocular surface is composed of the cornea, conjunctiva, lacrimal glands, meibomian glands, and other accessory glands.1 The eye and the ocular surface are also closely related to other organ systems. The nervous and endocrine systems control and affect the secretion of multiple glands on the ocular surface; immune cells migrate to the conjunctiva during infection and inflammation; the vascular system provides the necessary nutrients for the ocular surface, and is responsible for tear fluid metabolism.

The tear film, secretion product of the ocular surface, is fundamental for maintaining the smoothness and homeostasis of the ocular surface. The components of the tear film include a lipid layer and an aqueous layer with soluble mucins, which are mainly secreted by the meibomian glands, lacrimal gland, and conjunctival goblet cells, respectively. The conjunctiva is a connective, secretory tissue, starting from the corneoscleral limbus and covering the inner surface of the eyelids. It enables the free movement of the eye, provides immune surveillance, and secretes soluble mucins into the tear fluid.1 The conjunctival epithelium consists of stratified epithelial cells and goblet cells attached to a basement membrane. It can spontaneously begin wound healing upon injury.2 However, in cases with extensive ocular injuries, such as cicatricial pemphigoid, Stevens–Johnson syndrome, or severe chemical/thermal burns, fibrosis and wound contracture are very likely when the injuries are left untreated. Hence, an appropriate biomaterial is needed for optimal healing.34 When more than one part of the ocular surface is affected, conjunctival reconstruction is a prerequisite for successful ocular surface repair. Currently, two major types of biomaterials are used for conjunctival application: Tissue grafts or biomacromolecular materials. Promising results have been reported with both biomaterials in animal studies and clinical trials.

The dry eye represents a major ocular surface disease. According to the 2007 report of the International Dry Eye WorkShop, dry eye is a multifactorial disease and the symptoms include eye discomfort, visual disturbance, tear instability and, potentially, ocular surface damage.5 Based on data from the previous studies of dry eye epidemiology, about 4.91 million Americans above 50 years of age suffer from dry eye disease. In addition, there are tens of millions of people with less severe symptoms that can be triggered through contact with adverse factors, such as contact lens wear or extended use of visual display terminals.6 In general, dry eye may have a substantial impact on a patient's quality of life. In patients with severe dry eye, the disease affects even the common activities such as driving and reading. Dry eye can be either aqueous-deficient or evaporative, which results from problems with the aqueous or lipid layer of the tear film, respectively. Dry eye is further categorized into different subtypes, depending on the primary cause.5 Although goblet cell dysfunction is not a major cause, most of the dry eye patients have a significantly decreased number of goblet cells. Lack of mucin will further disturb the already sub-optimal tear film and ocular surface, increasing the severity of dry eye. Hence, while developing a treatment for dry eye, goblet cell repopulation should also be considered.

The most common management for a mild-to-moderate dry eye is tear supplementation. Artificial tears, ointments, and gels are examples of lubricants that protect the ocular surface and treat dry eye. However, some ingredients, especially the preservatives in these products, may damage the ocular surface epithelium if used as long-term. In patients with severe dry eye, surgical procedures, such as gland transplantation, is necessary to restore a healthy ocular surface and tear film. Over the past two decades, research has focused on evaluating the pathology of dry eye and developing models for in vitro and animal studies.7 Instead of simply hydrating the eye, future therapies will shift to strategies stimulating natural tear secretion to maintain ocular surface homeostasis, reducing inflammation, and inhibiting adverse effects.8 A robust in vitro model for normal human ocular surface and dry eye is also needed for drug screening purposes and pathophysiology studies.

The ideal material for conjunctival reconstruction should be a stable, thin, and elastic membrane, biocompatible with the human body.3 This review summarizes the major materials that have shown significant promise in either animal models or clinical trials. These materials include both autologous/allogeneic tissue products and biomacromolecular materials, and a novel collagen-based membrane that our group has been studying over the past few years, called as collagen vitrigel. This review will end with a discussion of developing an “organ-on-a-chip” as a potential therapeutic strategy for dry eye disease.

AUTOLOGOUS TISSUE GRAFTS

An autograft is a piece of tissue harvested from a healthy site in the same patient and transplanted to the damaged site to promote healing. As it is from the same individual, an autograft is the safest tissue product that can be used. In conjunctival reconstruction, autologous tissues are either conjunctival or nonconjunctival mucous membranes. A conjunctival graft from the same or contralateral eye is often applied following the excision of a pterygium, tumor, scar tissue, or other lesions. As the graft is usually small, the donor site is often left to heal spontaneously, or can be closed with a local flap.239 Studies have shown that conjunctival defects replaced with healthy conjunctival autografts have less wound contracture and lower risk of disease recurrence. An early study by Tan et al. found that donor conjunctival grafting reduced the recurrence rate of pterygium to <5%, compared to tissue excision alone, which had a recurrence rate of 60%.10 Although this procedure is effective, there are several drawbacks. First, autograft transplantation is limited to unilateral injury, as normal conjunctiva should exist in at least one eye to serve as the donor site. Therefore, autologous conjunctival transplantation is impossible in bilateral chemical/thermal burns or systemic ocular surface diseases. Other concerns with this procedure include postoperative discomfort and donor site morbidity, such as fibrosis.2

To overcome the limitations of autologous conjunctival grafts, mucous membranes from other areas in the patient have been developed as replacements. Oral and nasal mucosas are common alternatives when healthy conjunctiva is not available. Oral mucosas harvested from different places in the mouth have variable properties. Grafts from the hard palate are the thickest and, thus, contract the least, but they are difficult to obtain when compared to grafts from the buccal or labial regions.11 Researchers have also expanded mucosal epithelial cells on the oral mucosal graft for the treatment of complete limbal stem cell deficiency. This procedure is called as “cultured oral mucosal epithelial transplantation.”31213 However, unlike nasal mucosa, an oral mucosal graft does not contain goblet cells; therefore, it is less satisfactory in the situations of extreme dry eye with mucous and goblet cell deficiency, such as Sjögren's syndrome.3

AMNIOTIC MEMBRANE

Human amniotic membrane (AM) is a tissue product harvested from the inner layer of the placenta. It is composed of a single layer of epithelium, a thick basement membrane, and the underlying avascular stroma.14 Currently, AM is the most widely accepted conjunctival substitute.9 In the 1940s, a fetal membrane containing both amnion and chorion was transplanted for conjunctival reconstruction, but the success rate was low as a result of live cell inclusion.15 In 1997, the US Food and Drug Administration produced guidelines and surgical standards on the procurement, processing, and distribution of a tissue product, such as AM. Subsequently, various applications of AM have been reported in the literature yearly. Similar to autologous grafts, allogeneic AM can be used as a replacement for damaged conjunctival stromal tissue. For example, Acelagraft™ (Celgene Corp., Summit, NJ, USA) is a denuded AM that has been lyophilized and sterilized with γ-irradiation. Moreover, AM can also be used as a preventive patch/dressing for the ocular surface to reduce inflammation, scarring, and neovascularization, such as Prokera® (Bio-Tissue®, Doral, FL, USA), where AM is attached to a soft contact lens-sized conformer. The beneficial effect of AM could also be utilized as an extracellular matrix (ECM) extract. The product, AMX (Amniotic Membrane eXtract, Dr. Emiliano Ghinelli, Verona, Italy), is available in Europe for topical applications.16

The resemblance between AM and conjunctiva in the composition of basement membranes makes AM a successful substitute for the treatment of various ocular surface conditions, including chemical or thermal burns, conjunctival tumors, or recurrent pterygia.317 When processed properly and preserved, AM could promote rapid epithelialization, reduce inflammation and vascularization, and suppress fibrosis and pain.14 An analysis of the composition of AM ECM extract indicated that the anti-inflammatory action of AM is due to the presence of interleukin-10. In addition, AM supports nerve growth, which explains the near scarless healing after the AM transplantation.18

Although AM and related products hold great potential in ophthalmic applications, the availability, cost, and standardization of AM preparation remain problematic. In general, the variable composition and properties of AM, reflecting donor differences and nonstandardized preparation, could influence transplantation and result in inconsistent outcomes. In addition, the mechanism of AM's beneficial actions and its precise molecular composition require extensive studies.19 Most importantly, as AM is an allogeneic tissue product, donor-associated risk of infectious transmission cannot be completely eliminated because only limited disease screening tests are performed prior to harvesting AM.4 The optimal method for processing AM without compromising the therapeutic value is the topic of intense research.16

Numerous characteristics require evaluation for the selection of the type of tissue grafts for conjunctival reconstruction. For example, injuries in the fornix require the graft to contract less than a graft in the bulbar conjunctiva. In the extreme dry eye, nasal mucosa is superior to oral mucosa and AM, because it contains goblet cells.11 The features of each tissue graft material are summarized in Table 1.

Table 1

Comparison of different tissue graft materials for conjunctival reconstruction*

BIOMACROMOLECULAR MATERIALS

Since autologous/allogeneic graft transplantations have numerous limitations, advances in ophthalmic tissue engineering techniques have led scientists to develop new biomaterial matrices. Biomaterials engineered in a laboratory-based study on macromolecules, such as synthetic polymers and proteins, could have better properties for healing ocular diseases and injuries than traditional autologous/allogeneic tissue grafts. First, bioengineered macromolecular materials are tunable, in that their properties can be designed precisely to accommodate different requirements. For example, the degradation rate of poly (lactide-co-glycolide) (PLGA) can be changed by varying the ratio of lactide to glycolide. Furthermore, these biomaterials have much more consistent qualities, and their compositions have been extensively studied. Hence, these biomaterials present a lower risk for translational use. Some biomaterials can be modified to have different functional groups or motifs to selectively promote the growth of a certain type of cells, and drug-loaded particles can be embedded in the materials for a controlled release. Due to these advantages, biomacromolecular materials are a very promising alternative to tissue grafts for ocular surface reconstruction.

Although there is a significant body of literature on many biomaterials for conjunctival reconstruction, with some positive and promising results, none of the existing materials meets all the criteria for an optimal substrate for conjunctival repair. The degradable polymer matrices PLGA20 and poly(ε-caprolactone) (PCL)21 were tested in vitro and on animal models as candidates for conjunctival reconstruction. Lee et al. developed a porous PLGA matrix using a solvent-casting particulate leaching method that was also modified with collagen, hyaluronic acid, and human AM components. The modified PLGA matrix showed increased cell adhesion and growth in vitro. In a rabbit model of conjunctival injury, a PLGA graft significantly prevented conjunctival contracture and stromal scarring.20 However, due to the preparation process, the PLGA matrix was neither elastic nor transparent. Ang et al. prepared ultrathin PCL membranes by solvent-casting and biaxial stretching. The material formed was highly flexible and promoted the attachment and proliferation of conjunctival epithelial cells in vitro.21 However, it has not been tested on an animal model of ocular injury, and it may be difficult to integrate into the host tissue, and there may be induced scar formation due to its aligned fibrillar structure.

Natural protein molecules, such as collagen22 and keratin,23 were also tested in the animal models for conjunctival regeneration. Similar to the PLGA graft, the porous collagen-glycosaminoglycan matrix prevented scar formation in a rabbit conjunctival injury model.22 The disadvantage of this matrix is that it is not elastic or transparent. Keratin film has good mechanical properties, including elasticity and transparency, and it is very tunable depending on the preparation process.23 However, it may still be too stiff for ocular surface use, and the phenotypic development of epithelial cells on a keratin substrate remains unknown, as keratin is not a natural component of the conjunctiva.

COLLAGEN VITRIGEL

Type I collagen is the most abundant component in the ECM of the conjunctival stroma; therefore, it is one of the top choices for biomaterial engineers when designing substrates for the ocular surface. However, the applications are limited, because collagen hydrogels are opaque and composed of loosely packed collagen fibrils, while the collagen fibrils in the native conjunctival stroma are highly condensed and well organized.13 To improve the properties of collagen hydrogels, Takezawa et al. developed a Type I collagen hydrogel membrane using a three-step processing method: Gelation, vitrification, and rehydration.2425 Vitrification is a unique step that allows water in a hydrogel to evaporate in a controlled manner, during which, collagen fibrils reorganize and form crosslinks between each other [Figure 1a and b]. Hence, a normal opaque collagen hydrogel is transformed into a thin, elastic, and transparent membrane, and, most importantly, its fibril density increases tremendously. This material is called as “vitrigel” [Figure 1c]. Beginning with the original recipe, our group has studied collagen vitrigel extensively for optimization in ocular surface applications.

Figure 1

The preparation of collagen vitrigel and its appearance and structure. (a) Three-step preparation. (b) Crosslinking among collagen fibrils occurs during the vitrification process. (c) The final vitrigel is a transparent and thin membrane. (d) Transmission electron microscopy reveals the nanostructure of the vitrigel – the presence of densely and randomly packed collagen fibrils (adapted and modified from Guo et al.28 and Calderón-Colón et al.26 with permission from Elsevier)

Electron microscopy revealed that the nanoscale structure of vitrigel is a network of randomly aligned collagen fibrils [Figure 1d]. There are three variables in the vitrification process: Temperature, relative humidity (RH), and time. Calderón-Colón et al. systematically varied these variables and studied the properties of vitrigel under each condition to optimize the manufacturing process. Optical, mechanical, and thermal properties were measured and compared, as they are the most relevant for ocular surface application. Conclusively, vitrification occurring at 40°C and 40% RH for a week yields the optimal vitrigel that is highly transparent, mechanically strong, and elastic, and denatures at a temperature well above the core body temperature of humans.2627 As cell proliferation and differentiation largely depend upon the surrounding environment, Guo et al. studied the influence of nanostructure on cell morphology and phenotype by making vitrigels from different vitrification conditions.28 Although fibrillar density increased with increasing temperature and time, fibrillar diameter remained constant in all mature vitrigels. The biological effect was investigated by growing corneal keratocytes on different vitrigels. Due to the difference in nanoarchitecture (fibrillar density, bending, etc.,), keratocytes developed distinct morphologies.28 The optimal vitrigel was able to maintain the native characteristics of keratocytes under in vitro conditions.28 Vitrigel is an effective substrate for corneal keratocytes, it was successfully used in the reconstruction of corneal epithelium and endothelium, and it could preserve the stemness of limbal stem cells in vitro.29

Vitrigel was designed for ocular surface application and has a well-defined composition and controllable structure. It also closely imitates the architecture and properties of the native conjunctival stroma. Therefore, we utilized vitrigel as a tissue substitute for cell transplantation in conjunctival reconstruction. Vitrigel was first used as a culture substrate for conjunctival epithelium. A greater number of mucin 5 AC-positive goblet cells were observed on the optimal vitrigel compared to collagen hydrogel and tissue culture plates. In the animal study, vitrigel was used as an epithelial cell transplant carrier to repair a conjunctival defect in a rabbit model. The vitrigel graft accelerated re-epithelialization and prevented wound contracture and scarring.4 In addition, the vitrigel graft aided the repopulation of goblet cells in the wounded area, which has not been previously reported.4 Therefore, along with other bioengineered materials, vitrigel is a promising alternative as a tissue substitute in ocular surface applications.

ORGAN-ON-A-CHIP SYSTEM FOR OCULAR SURFACE AND DRY EYE

An organ-on-a-chip is a bioengineered microdevice with living cells, and it usually employs microfabrication and microfluidics technologies. It mimics the vital functions of the living organ and recapitulates organ-level pathophysiology in vitro.30 Organs-on-chips have great potential for studying the physiology of normal or diseased organs.31 The organ-on-a-chip technique emerged as a cell culture model to replace some costly and time-consuming animal studies in the pharmaceutical industry. Regular two- or three-dimensional cell culture was not completely successful because tissue development depends on both the architecture of the ECM and its mechanical/structural features.32 Due to the small size, the chip system can be used as a high-throughput drug-screening device, and it also has therapeutic potential. Theoretically, it could be engineered from dissected tissue, primary cells, or induced pluripotent stem cells.33 Over the past few years, several organ systems have been created on a chip device. A “lung on a chip” was created by opposing two poly (dimethylsiloxane) channels separated by a thin, porous, and flexible membrane. Human alveolar epithelial cells and pulmonary microvascular endothelial cells were cultured on opposite sides of the membrane. In the final product, air was introduced into the epithelial compartment to create an air-liquid interface and, together with the two side vacuum chambers; the microdevice was able to manipulate fluid flow and membrane stretching, as well as nutrient delivery.32 Liver, gut, and spleen organs-on-chips systems have also been developed.343536 Recently, bone marrow-on-a-chip, composed of artificial bone and living marrow, was first generated in mice and then maintained in vitro within a microfluidic device.37

Thus, such organs-on-chips may have a specific use in treating complex ocular diseases. For example, dry eye is a complicated disease affecting all parts in the ocular surface. Currently, dry eye therapeutics is tested in animal models before proceeding to clinical trials. However, the pathophysiology of dry eye has very different mechanisms in humans and in other species. The normal composition of tear fluid also varies among different species. Ophthalmic pharmaceutical companies are developing in vitro corneal epithelial models to evaluate the safety and efficacy of novel tear substitutes/lubricants. In vitro models of conjunctival epithelium and artificial lacrimal glands have also been investigated in the laboratory.3839 However, these models contained only a single cell type, and they could not be maintained for an adequate duration in vitro for physiological study. Hence, for future dry eye studies and drug development, it is critical to have an in vitro model that recapitulates the normal or diseased state of the human ocular surface. The combination of multiple cell types, such as conjunctival epithelium, lacrimal gland acinar cells, and microfluidic channels, will create a model of an aqueous tear-secreting unit. Although some difficulties exist, such as the necessary biological and mechanical cues for the construction of chips, advances in gene regulation and protein expression of the ocular surface have shed light on a potential design for “ocular surface-on-a-chip” for the study of dry eye. For example, these chips can be constructed with genetically modified cells to recreate a disease-state organ. Marko et al. discovered that a single gene knockout in a mouse model (SPDEF−/−) resulted in complete goblet cell loss and moderate dry eye, which is very similar to Sjögren's syndrome.40 Therefore, it would appear that ocular chips made from SPDEF−/− cells could serve as a comprehensive in vitro model for Sjögren's syndrome. Furthermore, linking a dry eye disease chip with a multi-organ microfluidic device that connects more than one single organ chip, such as chips reproducing the immune and vascular systems, would allow a greater understanding of the overall pathophysiology of dry eye.

SUMMARY

The ocular surface is the external protective component of the eye, and the conjunctiva plays an important role in this protective system. Researchers have been successful in developing tissue- and biomolecule-based grafts to reconstruct conjunctiva. However, improvement is required to render the available biomaterials more consistent, available, and biocompatible. Dry eye, which can be a serious ocular surface disease, affects tens of millions of people worldwide. While traditional treatment has major obstacles, breakthroughs in organ-on-a-chip technology offer new directions in establishing in vitro models of the ocular surface for dry eye disease research.

Financial support and sponsorship

King Khaled Eye Specialist Hospital-Wilmer Collaborative Research Grant; Research to Prevent Blindness Foundation.

Conflicts of interest

There are no conflicts of interest.